BRPI0705059B1 - hydrolysates of keratin, process for their production and cosmetic compositions containing the same - Google Patents

Abstract

keratin hydrolysates, processes for their production and cosmetic compositions containing them. The present invention relates to a process for the hydrolysis of keratin by microbiological and / or enzymatic processes. in particular keratin is derived from animal feathers such as chicken and is hydrolysed by a strain of badiius sp .. hydrolysates have a molecular weight of less than 500 da, making them ideal for cosmetic applications, in particular for applications in compositions for re-constructive hair fiber treatments.

Description

Keratin Hydrolysates, Process for Their Production and Cosmetic Compositions Containing the Same Field of the Invention The present invention relates to a process for keratin hydrolysis through microbiological and / or enzymatic processes. In particular, keratin is derived from animal feathers, such as chicken, and is hydrolyzed by a strain of Bacillus sp ..

Hydrolysates have a molecular weight of less than 500 Da, which makes them ideal for cosmetic applications, especially for composition applications for re-constructive hair fiber treatments.

Background of the Invention It is known that keratin has great value in the cosmetic industry for its action on hair fiber. Keratin hydrolysates may be prepared by acid or alkaline hydrolysis, or by enzymatic digestion. The function of chemical or enzymatic hydrolysis is to divide the peptide chains into smaller peptides with lower molecular weights. Commonly used starting keratin can have various origins: it can be derived from human hair fibers, wool, hair, feathers and horns. The prior art has several documents relating to keratin hydrolysates, and their use in cosmetics. US 7,220,405 discloses the formulation of peptide-based cosmetics such as body moisturizing conditioners and hair dyes, skin dyes and nail dyes. Peptides have been described as being able to bind with high affinity to hair, skin and nails. Thus this work aimed at the development of personal care products! containing peptides from collagen, elastin, soy, casein, silk and others, coupled to the active agent of the product directly or via a spacer.

This patent also uses hydrolysates of various proteins, showing the effectiveness of these peptides in cosmetics, but does not use hydrolysed feather keratin or the enzymatic method for hydrolysis of said proteins. US 4,186,188 describes the use of trypsin to hydrolyze proteins generally, generating positively charged 200 to 2000 Da polypeptides that can be used in cosmetic formulations for hair, nails and skin. Trypsin is a peptidase that has no action on keratin, so it cannot be used to hydrolyze keratin. In our work the keratin hydrolysis is made by keratin-acting keratinases and peptidases produced by B. subtilis AMR during the fermentation of chicken feathers. For this reason our enzymatic-microbiological method has a higher specificity, because keratinase acts directly on the keratin polymer.

US 2006/134092 and US 5,395,613 describe peptidases (enzymatic characterization work) produced by microorganisms of the genus Bacitlus and Micrococcus sedentarius capable of degrading proteins highly resistant to denaturation and degradation, including keratin, prion (first work) and collagen ( second work).

The above patents aim to describe a Bacillus or Micrococcus sedentarius peptidase for the degradation of difficult degradation proteins such as keratin. Our work makes use of another microorganism, Bacillus subtilis strain AMR keratinolytic, for the degradation of keratin. The purpose of our work was not to recover the enzyme but to use hydrolysis products for addition in cosmetic formulations. US 6,858,215 discloses a method for treating hyperkeratinized tissues in mammals employing proteolytic enzymes originally developed for the hydrolysis of food-associated proteins and is currently commonly used for tenderizing meat and improving the taste of food. The composition of the product developed in this patent has these softening enzymes, which soften and exfoliate hyper-keratinized skin formations such as calluses, granules, dryness, scaly skin and keratoses without harming adjacent tissues by selective lysis of hyper-keratinized tissues. The enzymes used (from 1 to 15% in the formulation) were Carlsberg subtilisin and a fungal Aspergillus oryzae peptidase.

This patent also describes a cosmetic and pharmaceutical product acting on a keratinized tissue. However, in this patent the enzymatic process aims at the hydrolysis of keratin "in situ", softening hyper-keratinized tissues such as corns. Our patent uses the feather keratin enzymatic hydrolysis product through the action of B. subíilis AMR peptidases and keratinases in formulation. Cosmetic for hair. The above authors' patent uses the enzymes directly on the skin. US 4,591,497 discloses an odor remover and deodorant which contains as a hydrolyzed effective component of keratin material (from 0.1 to 10% by weight of 200-5000 Da), of hair, feathers, nails, hooves, horns. and scales. Keratin hydrolysis was achieved by known methods using acids, alkalis or enzymes. The compound effectively acts on mercaptans and hydrogen sulfide by removing their odor. The use of keratin hydrolysates from the above patent has a different purpose than the one presented by our work. Obtaining this hydrolyzate also employs a different methodology. While the above work used acid, alkaline or enzymatic hydrolysis, we used enzymatic-microbiological hydrolysis.

US 5,262,307, CA 1108542 and US 4,390,525 describe pretreatment of keratin-containing material (feathers, hair, etc.) with a reducing agent or acid treatment, followed by enzymatic digestion of denatured keratin obtained in the first phase of process. In the first patent pretreatment is made with sulfite in aqueous solution (at 60-100 degrees) for denaturation of keratin. In the second, before enzymatic hydrolysis in the presence of urea the keratin is subjected to acidic conditions at a temperature of 80 ° C. The third patent describes the development of a keratin hydrolyzate containing at least two mercapto groups per molecule and having a molecular weight of 2,000 to 20,000 Da, suitable for cosmetic hair applications, in particular fixatives. The hydrolyzate is prepared by reducing the keratin in aqueous solution under alkaline conditions containing mercaptan and sulfite, followed by enzymatic hydrolysis. Oligopeptides achieved at the end of the described processes may be used in cosmetics.

In the cited patents enzymatic hydrolysis of previously reduced keratin is performed under alkaline conditions or in the presence of sulfite or partially hydrolysed in acid. In our work, hydrolysed keratin is obtained from microbial and enzymatic degradation of whole feathers without any prior chemical treatment.

Some patents that address keratin hydrolysis by physical methods are described below: US Patent 5,772,968 describes a system, including apparatus and methods, for hydrolyzing keratin-based materials such as unused carcasses, hair, feathers, and the like to obtain useful and marketable protein products. This 'hydrolysis system1' makes use of apparatus which promotes keratin breakdown by heating, expansion, stirring, mixing and drying. The high temperature added to the feather mixing process (keratinolytic material selected for the job) in a screw conductive tube leads to hydrolysis of the substrate. The goal of keratin hydrolysis is to increase its digestibility and that it can be used as an animal food supplement. US 4,172,073 describes keratin hydrolysis obtained from animal structures using high pressure saturated steam, finally obtaining a water soluble flour and excellent for use in animal feed as it is digestible by pepsin.

These patents describe physical processes (temperature, mechanical action, high pressure) to break the keratin molecule so that it can have its digestibility increased, that is, aiming at its food use. Our work proposes the microbial and enzymatic hydrolysis of feather keratin, in a process that does not require heating expenses, aiming to apply the small peptide fragments generated in hair cosmetics.

Some patents dealing with keratin hydrolysis by chemical methods are described below. US 2007/0065506 describes the use of keratin and its derivatives (50-60,000 Da) as an orally administered supplement for the reduction of oxidative stress and the benefits of this as promoting skin health and promoting anti-inflammatory response. The keratin derivatives produced were S-sulfonated keratin intermediate filaments, S-sulfonated high sulfur keratin and S-sulfonated hydrolyzed keratin peptides. Keratin derivatives were obtained from human hair, wool, animal fiber, hooves and horns, by partial oxidation. The keratin in this patent was obtained from mammals, while chicken feathers were used in our work. The treatment of keratin in the above patent was chemical while we used enzymatic-microbiological methods. The final product is also completely different. US 2007/128134 discloses a water soluble keratin derivative and its applications. The keratin derivative was obtained by the alkaline treatment of feathers followed by exposure to high energy wavelength irradiation, leading to molecular weight peptides ranging from 5000 to 50,000 Da. US 2004/0210039 reports a process for solubilizing keratin of keratin materials such as chicken feathers. The keratin was solubilized using sulfite under alkaline conditions. In this process the cysteine residues are partially modified by alkylation and the keratin is partially hydrolyzed. This partially hydrolyzed keratin, with molecular weight between 1,000 and 10,000 Da, can be used for the production of films. US 5,679,329 describes the development of a cosmetic composition containing milk protein (0.02% - 15%) and / or hydrolyzed milk protein and hydrolyzed keratin (0.1 - 10%) with molecular weight between 100 to 200,000 Da Hydrolyzed keratin can be obtained by hydrolysis of hair, wool, skin, silks, feathers, scales, hooves and horns. In this work the keratin used was derived from moderate acid hydrolysis (fragments of approximately 100,000 Da found in KERASOL marketed by CRODA) and controlled (fragments of approximately 150 Da obtained in CROGUAT WKP product also marketed by CRODA). The final product is preferably mousse used to fix hairstyles US Patent 5,154,916 describes the addition of 50,000 Da molecular weight keratin hydrolyzate in eyelash masks using a wax base. improving eyelash covering properties, stability and eyelash length. Hydrolysis keratin can be obtained from hair, wool, hooves, horns, hair, silks and feathers. Preferably hydrolyzed skin keratin was used by moderate alkaline hydrolysis at a concentration of 0.05 to 5%. U.S. Patent 4,839,168 describes the formulation of capillary cosmetics containing plant extract (preferably birch, rosemary and hamadrade) extracted with polar solvent and a polypeptide compound (weighing 100 to 100,000 Da) including keratin, keratin hydrolyzate, silk and hydrolyzed silk. The keratin used is derived from human hair, wool and feathers and has been extracted by oxidation and reduction methods and hydrolyzed by acid hydrolysis. The product aims to improve the sensory characteristics of hair. US 4,818,520 describes the use of keratin hydrolysates, obtained from keratinized material such as bird feathers, which may be useful in the formulation of products that reduce skin lines, moisturizers, skin masks, shampoo, shaving lotion, strengthening. of nails and conditioners. The hydrolyzate is obtained by heating refluxing feather meal in reflux, followed by cooling, filtration and acidification with hydrochloric acid and re-filtration, heating and evaporation. Finally a new alkaline treatment step is made followed by neutralization and so on. you get liquid hydrolyzed keratin.

US 4,465,664 and US 4,460,566 describe the composition of hair products containing at least one type of keratin-based material derivative, such as animal hair, human hair, wool, nails, feathers, hooves, horns and the like. Keratin derivatives were obtained by oxidation (which converts disulfide bridges to sulfonic acid) or reduction (which produces derivatives with thiol groups) of keratin and used at the concentration of 0.01-10% and 01-10%, respectively, and size between 30,000 to 100,000 Da. The first patent product has a moisturizing effect on the hair. US 3,970,614 discloses solubilization of keratin-based material such as chicken, hair, hair and hoof feathers by treatment with high temperature N, N-methylformamide to produce a protein hydrolyzate suitable for use as an animal and human food supplement. and as a raw material in the food industry. The main difference between the patents described above and the methodology developed by our group is the process used for keratin hydrolysis, while the patents described above use chemical hydrolysis (acid and / or alkaline, or using N, N-dimethylformamide), or oxidation and reduction reactions, ours uses enzymatic-microbial hydrolysis, and the source of keratin is also different in some cases. While some of these works use keratin from bovine hooves and skin, we use chicken feather keratin. Our product uses only hydrolyzed keratin with a molecular weight of less than 500 Da, which facilitates the adsorption and penetration of small peptide fragments into the hair fiber. The final product also has different purpose from some of the above patents. Roddick's article (Roddick-Lanzilotta, A & Kelly R. 2006. Protecting the Hair with Natural Keratin Biopolymers. Cosmetics & Toiletries, 121 (5)) describes two strategies for producing intact keratin biopolymers and wool keratin peptides of sheep for application in hair cosmetics. This polymer binds to preferred sites on the fiber, protecting hair from damage, and contains antioxidant properties. Keratin production involves purification steps to obtain intact keratin (55 kDa), which has film-forming properties that can be applied to cosmetics. Low molecular weight peptides, capable of entering the hair fiber cortex, were obtained from Croda Chemicals Europe, ie obtained by chemical hydrolysis. Obtaining the intact keratin cited in the above work was not the target of our work, although intact feather keratin (approximately 10 kDa) was found in the culture supernatant of the microorganism used (data not shown). The article cites purification steps which burden protein production. The hydrolysate used by them was obtained by chemical hydrolysis, while that of our work was by microbial hydrolysis.

In the literature there is a large amount of work reporting feather degradation by keratinolytic microorganisms through submerged fermentation of feathers aiming to use the keratin hydrolyzate obtained as a dietary protein supplement in animal diet. In contrast, our work aimed to apply these hydrolysates from microbial digestion. in cosmetic formulations for hair use, an unprecedented purpose for these feather fermentation products. Listed below are patents, theses and scientific articles using microbiological feather hydrolysis to obtain keratin hydrolysates for use in feed. None of the documents cited below use the microorganism Bacillus subtilis strain AMR

The principal documents are US 6,214,576, US 5,887,000, US 5,186,961, US 5,063,161 and US 4,908,220.

Maciel, J. L. Production of protein hydrolysates from chicken feathers using keratinolytic bacteria. 2006. Master Thesis. UFRGS.

Grazziotin A., Pimentel, F.A., Sangali, S., Jong, E.V. and Brandelli, A 2007. Production of feather protein hydrolysate by keratinolytic bacterium Vibrio sp. kr2. Bioresour, Technol. 98 (16): 3172-5.

Geun-Tae Parka, Flong-Joo Sonb, 2007. Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. Microbiological Research (in press).

Mio Kojima, M., Kanai. M., Tominaga, M., Kitazume, S., Inoue, A. and Horikoshi, K. 2006 Isolation and characterization of a feather-degrading enzyme from Bacillus pseudofirmus FA 30-01. Extremophiles 10: 229-235.

Brandelli, A. & Riffel, A. 2005. Production of an excellular keratinase from Chryseobacterium sp. growing on raw feathers. Electronic Journal of Biotechnology, ISSN: 0717-3458.

El-Refai, Η. A, AbdeINaby, Μ. A., Gaballa, A., El-Araby, Μ. H. & Abdel Fattah, A. F. 2005. Improvement of the newly isolated Bacillus pumilus FH9 keratinolytic activity. Process Biochemistry, 40: 2325-2332.

Grazziotin, A., Pimentel FA, de Jong, and V and Brandelli A. 2005. Nutritional improvement of feather protein by treatment with microbial keratinase. Animal Feed Science and Technology, article in press Suntornsuk, W. & Suntornsuk, L 2003 Feather degradation by Bacillus sp. FK46 in submerged cultivation. Bioresource Technologyy, 86: 239-243.

Nam, G., Lee, D., Lee H, Lee, N., Kim, B., Choe, E., Flwang, J., Suhartono, Μ. T, and Pyun. Y. 2002.

Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe Arch Microbiol 178: 538-547.

Longshaw, C.M., Wright. J. D., Farrel, A. M. & Holland, K. T. 2002. Kyptococus sedentarius, the organism associated with pitted keratolysis, produces two keratin-degrating enzymes. Journal of Applied Microbio 93: 810-816.

Muhsin, T.M., & Hadi, R.B. 2002. Degradation of keratin substrates by fungi isolated from sewage sludge Mycopathology 154: 185-189.

Yamamura, S., Yasutaka, M., Quamrul, H., Yokoyama, K. & Tamiya, E. 2002. Keratin degradation: a cooperative action of two enzymes from Stenetrophomonas sp. Biochemical and Biophysical Research Communications, 294: 1138-1143.

Kim, J.M., Lim, W.J., Suh, H.J. 2001. Feather-degrading Bacillus species from poutry waste. Process Biochemistry, 37: 287-291 el-Naghy, Μ. A., El-Ktatny, M.S., Fadi-Allah, E.M. & Nacer, W.W. 1998. Degradation of chicken feathers by Chrysosporium georgiae. Mycopathology, 14 (2): 77-84.

Dalev, P., Ivanov, I. & Liubomirova. 1997. Enzymic modification of feather keratin hydrolysates with lysine aimed at increasing the biological value. J. Sci Food Agric, 73: 242-244.

Singh, C. J. 1997. Characterization of an extracellular keratinase of Trichophyton simii and its pole in keratin degradation. Mycopathology, 137: 13-16.

Santos, R. M. D. B., Firmino, A. A. P. & Felix, C. R.1996. Keratinolytic activity of Aspergillus fumigatus fresenius. Curr Microbiol, 33 (6): 340-370.

Lin, X., Lee, C., Casale, E, S. & Shih, J. H. 1992, Purification and characterization of a feather-degrading Bacillus lichemformis strain. Applied and Environmenial Microbiology, 58. 3271-32575 OBJECTS OF THE INVENTION An object of the present invention is a keratin hydrolysis process comprising the steps of: a) mixing: a.1) from 106 to 5x10 5 cells / ml of at least a bacterium of the species B.subtilis: a.2) from 0.1% to 5% w / w of a keratin fiber containing material; a.3) from 0.005% to 10% w / w of culture medium; where the pH of the medium is in the range from 7.5 to 8.5; b) keeping the mixture in a) at a temperature ranging from 20 ° C to 35 ° C and under stirring, c) separating the supernatant obtained in b); d) separating the hydrolyzed peptides from the supernatant from step c);

In a preferred embodiment of the invention, B. subtilis is B.subtilis strain AMR

In a preferred embodiment of the invention, the keratin fiber-containing material is chicken feather.

In a preferred embodiment of the present invention, the pH is 8.0.

In a preferred embodiment of the present invention the culture medium comprises yeast extract, peptone, KCI, sucrose or a mixture thereof.

In a preferred embodiment of the present invention, the mixture is maintained at a temperature of 28 ° C and under stirring of 300 rpm.

In a preferred embodiment of the present invention, separation of the supernatant is done by centrifugation, especially by centrifugation at 4,000 rpm for 20 min.

In a preferred embodiment of the present invention, the separation of hydrolysed peptides is by ultrafiltration. A further object of the present invention is an optional process for preparing keratin fiber-containing material comprising at least one washing and / or delipidation step.

In a preferred embodiment, the chicken feathers are washed with detergent and water. dried, delipidated with a mixture of chloroform methanot and dried. A further object of the present invention is a keratin hydrolyzate having a molecular weight ranging from 500 to 1000 Da. In particular, the keratin hydrolyzate is obtained by the process of the present invention. A further object of the present invention is a cosmetic composition comprising a keratin hydrolyzate in a concentration ranging from 0.0001% w / w to 20% w / v.

In a preferred embodiment of the present invention, the cosmetic composition is applied to keratinous tissues such as hair, skin and / or nails.

In a preferred embodiment of the present invention, the cosmetic composition is a shampoo.

In a preferred embodiment of the present invention, the cosmetic composition is a conditioning cream.

Description of the Figures Figure 1 shows the MALDI-TOF of B. subtilis culture supernatant, AMR in feather medium after 120h culture, showing peptides derived from feather keratin. Figure 2 shows the MALDI TOF of CRODA keratin hydrolysates obtained from pig's hooves. Figure 3 shows the gelatin and keratin zymography of feathered Bacillus subtilis supernatant demonstrating the presence of proteolytic and keratinolytic enzymes. Figure 4 shows the HPTLC of feather keratin hydrotisates by microbial degradation. 1- glycine control; 2-peptides collected by the 1000 Da membrane; 3-peptides collected by the 500 Da membrane; Commercial 4-hydrolyzate Queratan. Figure 5 shows the SDS-PAGE of the 1000 Da membrane retained and filtered material by the AMICON ultrafiltration process. Staining: silver nitrate Figure 6 shows the zymogram of material retained and filtered by the 1000 Da membrane by the AMICON ultrafiltration process. Coloring: coomassie blue. Figure 7 shows the flowchart of the methodology employed, exemplifying the treatment to which the locks were submitted. Figure 8 shows the evaluation of the hydration content for the heatless dry swabs (room temperature). Group of strands: 1 - virgin hair; 2 - dyed hair; 3 - dyed hair with lights; 4 - dyed hair with straightening; and 5 - totally discolored hair. Figure 9 shows the hydration content assessment for the heat-dried slabs and thermal plate. Group of strands: 1 - virgin hair; 2-dyed hair; 3 - dyed hair with lights; 4 - dyed hair with straightening; and 5 - totally discolored hair. Figure 10 shows the graphical representation of the sensory analysis results for the heatless dry wick group. Figure 11 shows the graphical representation of the sensory analysis results for the heat-dried wick group.

Detailed Description of the Invention The examples described herein are not intended to limit the scope of the invention, but merely to exemplify a preferred embodiment of the invention. Any other similar and / or equivalent embodiment should be construed as falling within the scope of the invention.

Keratin Fiber Containing Material A suitable keratin fiber containing material according to the present invention may be selected from the group comprising natural sources of keratin fibers such as hair, feathers, hooves, nails, horns and the like. Feathers are a preferred starting material, especially chicken, chicken, ducks, geese or other birds, such as by-products of the poultry industry. The keratin fiber is preferably subjected to pretreatment such as cleaning, washing, delipidating, cutting, milling, drying or combinations thereof. This pretreatment is intended to facilitate keratin fiber handling and may improve overall process efficiency and may also influence the quality of the final product. B. subtilis The bacterium useful in the present invention is the Bacillus subtilis species. In particular, the species useful in the present invention may be chosen from several variants. The most suitable variant is B. subtilis strain AMR.

Cosmetic Composition A person skilled in the art can choose the appropriate form, as well as the method of preparation, based on knowledge of the field, taking into account the nature of the ingredients used, in particular their solubility, and also the intended use of the composition.

The compositions according to the present invention may be in the form of aqueous solutions, alcohol-water, or oily mixtures, oil-in-water, water-in-oil or multiple emulsions, aqueous or oily gels, anhydrous products, or dispersions. of an oil phase in an aqueous phase comprising nanoparticles, such as nanospheres and nanocapsules, or ionic (liposome) and / or nonionic lipid vesicles.

When the composition according to the invention is an emulsion, the proportion of the oil phase may vary, for example from 5% to 80% by weight, preferably from 5% to 50% by total weight of the composition. The oils, emulsifiers and co-emulsifiers used in the composition are chosen from those conventionally used in the corresponding technical field. The emulsifier and co-emulsifier may be present in the composition in a ratio ranging from 0.3% to 30% by weight. preferably from 0.5% to 20% by weight relative to the total weight of the composition.

For hair applications, the composition may preferably be in the form of creams, lotions, gels, emulsions or mousses, or as aerosols comprising a pressurized propellant. It may be in the form of a hair care lotion, a shampoo or conditioner, a liquid and / or solid soap, a hair styling product (styling gel, mousse, hairspray), a coloring shampoo, a straightening make-up, a Sparkling cream. It may also be in the form of a dye to be applied by brush or comb.

For nail applications, the composition may preferably be a product such as a colored nail polish, a base nail polish, or other product to be applied under or over another product, a nail polish remover, or a product to protect, strengthen and / or repair. the nails.

For skin applications, the composition may preferably be more or less viscous, and may have the appearance of, for example, a white or colored cream, an ointment, a lotion, a paste.

The compositions may be applied by any suitable means, such as brushes, sprays, or fingers, for example.

The compositions are also associated with processes for care, treatment, strengthening and / or repair of keratin substrates, wherein the composition defined in the present invention is applied to the skin, hair (including eyelashes) and / or nails, optionally followed by rinsing.

For the purposes of this invention the term "keratin substrate care" means a composition directed to improving the appearance and / or surface of keratin substrates. Care of such substrates may consist of making hair softer and less brittle.

For the purposes of this invention the term "strengthening and / or repairing keratin substrates" means a composition intended to conserve and / or restore the physical and / or mechanical properties of such substrates, which may manifest as, for example: stiffness of keratin substrates which It gives greater consistency and also a more pleasant touch, resulting in an increase in the volume of keratin fibers and also easy styling and maintenance of the hairstyle; better elasticity and / or resistance to applied mechanical forces, such as during combing. The keratin hydrolyzate obtained does not interfere with the color of the product to which it is applied (shampoo and rinse cream) as it has a dare color, unlike many commercially available hydrolysates that have a strong hue and may influence the final color of the product. The hydrolysate compound of the present invention may be present in the cosmetic formulation in a ratio of 0.0001% w / w to 20% w / w.

Main Advantages The main advantages of the hydrolyzate presented in this invention in relation to the state of the art are: - Obtaining peptides of lower molecular weight; The use of 500 Dalton molecular weight peptides allows better penetration of the peptides into the hair cuticle. Commercially available preparations have peptides in the range of 980-1300 Daltons. - Use of clean technology Enzyme biocatalyzed processes are less polluting. The characteristics of these industrial processes and the biodegradability presented by their effluents meet the requirements of ISO 9000 and ISO 14001, which set standards for product quality and guide the characteristics of production processes, emphasizing lower energy consumption, low impact. environmental and product quality. In this context it is important to emphasize that enzymatic processing of raw materials results in higher value added products. - The use of microorganism as direct producers of biocatalyst (enzyme) in the production system of keratin hydrolysates. The use of microorganisms in the production systems of keratin hydrolysates is advantageous because large populations of microorganisms and consequently more enzyme can be easily obtained, reducing feather degradation time, and having a production control at all times. phases. Other advantages are that microorganisms can be genetically improved by increasing the quantity and / or quality of the biocatalyst produced, also allowing always to select high activity strains - Growth medium is cheap Growth medium is cheap due to the abundance of raw material in the Brazil (agro-industrial waste from the poultry industry) Therefore, it is a method that still has the advantage of potentially being able to take advantage of one of the main waste generated by Brazilian industrial activities. - Favors the recycling of materials and can generate productive agreements between companies. The cosmetic industry can make an important link with the poultry industry to take advantage of these residues, preventing them from being incorporated into animal feed or polluting the environment. The composting system that the industry uses of chicken feathers and bedding is not feasible to absorb all domestic production.

Example 1. Preparation of Chicken Feathers Feathers used in the culture medium were detergent-washed white chicken feathers in running water, dried at 60 ° C and delipidated with chloroform: methanol (1: 1 v / v) for 1h while stirring. 300 rpm at room temperature. Delipidation was performed in a 4 liter Becker with 1/3 of the volume in feathers with 1 L of chloroform: methanol solution 1; 1 (v / v). Then the feathers were removed and dried overnight at 60 ° C. Feathers were added whole in the culture medium as the main source of carbon and nitrogen.

Example 2. Micro-culture and culture conditions To obtain the keratin hydrolysates Bacillus subtilis cepa AMR was used, isolated by our RICA poultry industry agro-industrial waste laboratory and currently deposited in the Oswaldo Cruz Foundation culture collection under the number 1266. This microorganism was chosen because of its intense keratinolytic activity for chicken feathers. The bacillus was grown in yeast extract medium (0.5% yeast extract, 0.5% peptone, 2.0% KCI and 2.0% sucrose;) for 2 days at 28 ° C under constant agitation (300 rpm ) to obtain cell mass and washed with saline (2x 3000rpm / 20min) for removal of the media components before being inoculated into the medium containing 1% feathers. Then the cells were transferred to PBS pH 8.0 medium (NaH2P04 0, 06M and 0.04M K2HP04) with 1% chicken feathers (prepared according to the procedure described in the previous item) and supplemented with 0.01% yeast extract. The sample was grown in this medium for 5 days at 20 ° C under 300 rpm agitation. At the end of bacillus fermentation in the feather-containing medium as the main source of carbon and nitrogen, the supernatant was collected by centrifugation at 4,000 rpm for 20 minutes (MAZOTTO, AM 2005 Bacillus licheniformis AMR keratinases isolated from the poultry industry. 2005. Monographic work ( Bachelor of Microbiology and Immunology) - Institute of Microbiology Professor Paulo de Góes / UFRJ, Rio de Janeiro, 2005).

Example 3. Analysis of peptides present in culture medium by MALDI-TOF

Matrix assisted laser desorption / ionization - Time of flight (MALDI-TOF) analyzes were performed to detect peptides in the culture supernatant (obtained according to the previous item) generated by feather hydrolysis by B.subtilis AMR peptidases. The supernatant containing 4.99 mg / m 2! of proteins dosed by the Lowry method (LOWRY, OH; ROSEMBROUGH, N.J; FARR, AL AND RANDALL.RJ.1951 Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 193 (1): 267 - 275), was partially purified on ZipTip C18. ZipTip C-iS was equilibrated with a 100% acetonitrile (ACN) solution followed by washing with 0.1% trifloroacetic acid (TFA). After this process, the peptides were fixed to the ZipTip C18 resin, washed. with 0.1% TFA for removal of salts, phosphates and / or DMSO that cause reading noise. Elution was done with 0.1% TFA in 50% ACN. Purified samples were incorporated into the 1: 1 α-cyano-4-hydroxycinnamic acid matrices (5 pg / mL in 0.1% TFA in 50% ACN). The mixture was then applied to the plate for analysis by MALDI-TOF. MALDI-TOF analysis revealed 800-1100 Dalton molecular weight fragments in the crude culture supernatant (Figure 1) (Mazotto, 2005). The same preparation procedure performed for the culture supernatant was also done for commercial preparation! of diluted CRODA 100x. The profile of small peptide fragments observed in the bacillus culture supernatant was also observed in the commercial preparation of CRODA (Figure 2) (Mazotto, 2005).

Example 4 Separation of keratin hydrolysates from chicken feathers and analysis of HPTLC-generated amino acids and peptides. The culture supernatant, besides containing the peptides derived from the feather hydrolysis, also had the keratinases and peptidases secreted by Bacillus subtilis AMR. Figure 3 shows two zymograms with gelatin and keratin of the 20X concentrated crude culture supernatant on overnight dialysis membrane against polyethylene glycol. Zymographs were prepared according to the methodology described by Heussen & Dowdle (HEUSSEN, C. & DOWDLE, E. B 1980. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulphate and copolymerized substrates. Anal. Biochem, 102: 196-202. ), but using gelatin (Merck) and keratin extracted from chicken feathers with DMSO as substrates incorporated into the 12.5% polyacrylamide mesh.

To have a peptide-only preparation, the supernatant was ultrafiltrated using the AMICON system to separate peptides up to 500 and 1000 Dalton through 500 and 1000 NMWL (Nominal Molecular Weight) Millipore regenerated cellulose membranes. Limit), respectively. Figure 4 shows these peptides and amino acids and compares them with a commercial preparation of POLYTECHNO hydrolysed feather keratin called Queratan, by HPTLC (high performance thin layer chromatography) and developed with ninhydrin, showing that feather hydrolyzed by B Subtilis AMR has a similar amino acid and peptide pattern to the commercial preparation used. Queratan also has, in addition, higher molecular weight peptides (Fig.6, line 5). Comparatively our procedure is better because it has only low molecular weight peptides that have better penetration power into the hair fiber. As a control, a glycine solution (1 mg / ml), an amino acid present in high concentration in the feathers, 76.6 g / kg (DALEV, P. 2000) was used. Use of biodegradable keratin and collagen containing wastes through enzymatic treatment. Wolfsburg, Orbiti Association).

In the HPTLC 10 µl of peptides separated from the 500 and 1000 Da membrane supernatant were applied.

During ultrafiltration, mainly proteins with molecular masses above their exclusion threshold were retained in the membranes, including enzymes and solubilized but not hydrolysed keratin molecules (with a molecular weight of approximately 10 kDa). Membrane retention and peptides smaller than their exclusion (filtered) limit were examined by SDS-PAGE (LAEMMLI, V.K 1970 Cleavage of structural during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. ) and stained by the silver nitrate staining method (Figure 5) and zymography (HEUSSEN. C. & DOWDLE, EB 1980. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulphate and copolymerized substrates Anal. Biochem, 102: 196 -202), as shown in Figure 6. The SDS-PAGE showed the presence of proteins only in the membrane retained (30 μΙ_ sample was applied), an expected result, since peptides and amino acids (60 pl of sample were applied ) are outside the detection range of the technique, the same is true for zymography. This showed that there was loss of culture supernatant activity during the ultrafiltration process as compared to the supernatant zymography prior to ultrafiltration (Figure 3 and Figure 6). SDS-PAGE and zymography showed the complete absence of proteins in the peptide fraction confirming that there was no contamination of the hydrolyzate with proteins and proteolytic enzymes respectively.

Example 5. Cosmetic Application of Chicken Feather Hydrolyzate Peptides Molecular weight peptides of less than 500 Dalton obtained by ultrafiltration on AMICON (500 NMWL membrane) were incorporated into two cosmetic bases: a mild shampoo and a rinse rinse cream. both containing 10% of the material filtered through 500 NMWL AMICON membrane

Gentle shampoo with 10% chicken feather microbial hydrolyzate containing peptides less than 500 Da Lauril sodium sulfate ether ............................ 30% Polyglycoside Decyl .................................... 5% Polyglycoside Lauryl ..... .............................. 5% Surfax acid .............. .. ................... 3% Coconut fatty acid diethanolamide ............ 4% Phenoc-hen or Phenova .... .................. 0.5% Germai 115 ................... ... ............... 0.2% Keratin Hydroisate ........................ 10% Unistab 569 .................................... 0.2% Fennel essence ..... ...................... 0.5% Distilled water qsp ..................... ...... 100 ml Germai requires heating (80 ° C) for its dissolution in distilled water. After dissolution of Germai, the remaining components were added to the solution. The polyglycosides must be heated for incorporation into the above solution. Homogenization should be done slowly to avoid foaming. Finish by adding the Microbial Feather Hydroisate, Unistab 569 and Essence, complete the final volume by homogenizing Rinse Rinse Cream with 10% Chicken Feather Microbial Hydrolyzate containing peptides less than 500 FROM OIL PHASE Cetostearyl Alcohol ..... ......... 5% Phenova ............................. 0.5% Cetyltrimethylammonium chloride .. .... 0.5% WATER PHASE

Germai 115 .......................... 0.2% Fennel Essence .............. .... 0.5% Dye ............................. 0.2% Keratin Hydroisate ..... ....... 10% Distilled water qsp ............... 100ml All components of the oil phase were heated (75 ° C) together and homogenized until complete dissolution. In another container the germ was dissolved under heating at 80 ° C. After reaching the temperatures, the oil phase was added to the aqueous phase under stirring until emulsified. The obtained solution was placed in a cold bath, homogenized and added to the other aqueous phase substances.

These products were applied to virgin and chemically treated locks, previously washed and degreased and for 5 weeks, subjected to treatment with mild shampoo and rinse cream, containing microbial keratin hydrolyzate, and heat drying followed by thermal plate. 180 ° C and room temperature to evaluate the degree of hydration of the hair fibers.

The locks were separated into 5 different groups. These are composed of virgin, dyed, straightened, light-dyed (bleached) and fully decayed strands of hair. Each group contains four strands, two with 10% keratin hydrolysates and two control strands. Prior to the performance of the tests, all locks were properly washed with 2% sodium lauryl sulfate shampoo. and rinsed with distilled water. This procedure aimed to eliminate any materials adsorbed to the wire, avoiding interference in the assay. Shown below is the flowchart of the methodology used for the hydration treatment and measurement process (Figure 7).

The hydration measurement tests were performed in a CORNEOMETHER brand CM 825 apparatus. In the same group of strands, control tests were performed with the cosmetic bases without the chicken feather hydrolyzate peptides. The formulations were manipulated at the UNESA Pharmacy Laboratory - Campus Rebouças. Sensory test was applied to evaluate hair shine and emollience.

Example 6. Hydration Measurement The hydration measurement process of the fuses was performed every seven days over a period of five weeks at the Galenic Development Laboratory (LADEG) of the UFRJ University Pharmacy. For this, we used the device CORNEOMETHER brand CM 825. The function of this device is to evaluate the amount of water evaporated through the hair fiber by field variation. The method is based on the large vapor variability of the water dielectric constant which is automatically recorded in the apparatus. The measured value is given in Arbitrary Hydration Unit (UA), being used for the measurement of hair strands. The temperature and humidity of the environment were always monitored and they were around 25 ° C and 42% humidity, respectively. .

For each group, 5 hydration measurements were made and the average of each evaluation was used to present and discuss the results. The ANOVA analysis of variance was used to statistically evaluate the final hydration content in the capillary structure with hydrolysed peptides of chicken feather keratin, in relation to the formulation without the hydrolyzate ie control group.

After the hydration treatments between the different types of hair, the following hydration degrees were observed: (table 1) Table 1: Moisture content obtained by each hair type. A - mecha zero point (washed only with sodium lauryl sulfate) and dry without heat (naturally); B - shampoo washed with 10% protein hydrolyzed peptide conditioner and dried with heat and flat iron; C - shampoo washed with shampoo and 10% protein hydrolysed peptide conditioner, and dried without heat (naturally); D - control mecha (washed with shampoo and conditioner control) dry with heat and flat iron; E - Control rinse (shampooed and control conditioner) dries without heat (naturally). These average results were obtained at the end of the five weeks.

Figures 10 and 11 represent the average results of the hydration evaluation of the heat-free, heat-treated, and thermal plate-dried wick group (figure 9). For both situations, comparisons were made between the hydration content of the control, zero point and 10% protein hydrolyzed peptide.

It was observed that the group of rinds treated with control preparation did not present significant hydration as those treated with 10% chicken feather protein hydrolysed peptide. The average hydration obtained by the control formulation treated group during the two evaluations (drying with heat and without heat) was lower than the group treated with 10% hydrolysed peptide. Even the locks that were subjected to heat obtained a lower degree of hydration than those that naturally dried at room temperature. This data confirms that the base used in the formulation did not interfere with hydration. Thus, it becomes clear the importance of the peptide components (hydrolyzed keratin) in the capillary structure, providing a greater degree of hydration. This is due to the role of these components in protecting, enriching or repairing hair fibers and making them more hydrated, as the amino acid and polypeptide fragments of hydrolyzed proteins that are composed of an amino acid group and have a low molecular weight. , appear to bind to hair keratin, restoring damaged protein structure (PAOLA, MVRV Ethnic Hair. COSMETICS & TOILETRIES, Sao Paulo, Brazil, v. 11, p. 36-44, May / Jun 1999).

For the statistical analysis of the results, the ANOVA analysis of variance method was used, from which it was possible to observe that the comparisons between the zero-point fuses and the heat-dried and heat-dried control fuses naturally (room temperature ) did not show significant results at the 5% level, nor did it show significant results, the fuses with the naturally dried peptide hydrolyzate without heat. However, the wicks that used the 10% protein hydrolyzed peptide had a significant hydration content of 5%. This more consistently confirms the results obtained previously through the averaging method.

Through the results it was observed that the virgin hair with its cuticle intact obtained a higher degree of hydration, since it allows the product to remain in its structure. Regarding chemically treated hair, it was observed that the absorption content is higher the lower the degree of aggression in its hair structure (EXPOSIT, F. Trichology. Spain, Madrid, ed. Eldesa, 5Q-58p, 1995).

Bleaching or treating hair with perming products decreases the amount of hair fiber cystine. Dramatic discolourations also lead to loss of tyrosine and methionine (SCANAVEZ, C. 2001. Alteration in hair care induced ultrastructure and its effects on color properties. PhD thesis under the guidance of porfa. Inês Joekes Institute of Chemistry - UNICAMP .) The use of products with keratin hydrolysates can minimize this loss and improve hair characteristics. The measurement method used is to verify the degree of hydration of the skin surface and how the evaluation was made to verify the degree of hydration of the nail. There may have been some interference in the results, since the capillary structure presents some differences in relation to the skin, such as a lower water degree (PEYREFITTE, G .; MARTINI, M .; CHIVOT, M. Skin Aesthetics-Cosmetics; Cosmetology; Gerai Biology, São Paulo: Andrei Organization Ltda, 373-379p, 1998. BOTELHO, A, J. Hydration Evaluation apilar on straightened hair using cosmetic preparation with seaweed extract. 2006. 61 f. Monographic work (Pharmacy Degree) - Estácio de Sá University, Rio de Janeiro, 2006). To confirm the results obtained, it would be necessary to perform the analysis of the capillary structure through electron microscopy.

Example 7, Sensory Analysis A test for sensory evaluation of the wick was also performed to check the degree of gloss and softness.

The evaluators (total of 20 people) analyzed the fuses (zero point) with the control ones and the tests, to verify which one had higher brightness and emollience. The evaluation was blind, as the evaluators did not know which formulation was being used. Before the test was performed, the evaluators underwent a hand hygiene process to remove any greasy product that could interfere with the analysis, being answered which group had greater or lesser degree of gloss and emollience.

Due to the sensory evaluation carried out to verify which of the locks (with 10% hydrolysate and control) presented higher brightness and emollience, different results were obtained according to the type of drying performed. For the group without dry fuses (at room temperature) it was observed that the highest preference percentage of the evaluators (65%) was for the fuses that had been treated with 10% protein hydrolysates and a smaller one (35%). It was not able to choose which of the groups had the highest brightness and emollience (Figure 10). On the other hand, for the group of heat-dried and thermal-plate fuses it was observed that the highest percentage of preference of the evaluators (90%) was for the fuses that had been treated with 10% peptide hydrolyzate and a smaller one (10%). It was not able to choose which of the groups had the highest brightness and emollience (Figure 11). These results were crucial to evaluate how much the use of protein hydrolyzed peptide interfered in the capillary structure making it more silky and shiny.

Figures 14, 15 and 16 show two groups of heat treated (chemically treated) control hair.

In the consumer's view the shine is directly related to healthy hair, because the higher the shine the healthier the hair will be (SCHUELLER, R .; ROMANOWSKI. P. Hair Brightness Assessment. Cosmetics & Toiletries, Melrose Park IL, United States States, v.14, pp. 52-56, Sep / Oct. 2002).

When using moisturizing cosmetics on hair, they are responsible for increasing the amount of water in the hair or forming a barrier that makes it difficult to lose water, because the moisture content of hair influences physical properties such as static load, stiffness, shine and volume (POZEBON, D. Hair analysis: a review of procedures for determining trace elements and applications. Química Nova, Florianópolis, v. 22, n. 6, p. 830-840, 1999. BOTELHO, A, J Assessment of capillary hydration in straightened hair using cosmetic preparation with seaweed extract 2006. 61 F. Work monograph (Undergraduate Pharmacy) - Estácio de Sá University, Rio de Janeiro, 2006).

Claims (17)

1 . Keratin hydrolysis process comprising the steps of: a) mixing: a.l) from 106 to 5x10 7 cells / ml of at least one bacterium of the species B.subtilis cepa AMR; a.2) from 0.1% to 5% w / w of a material containing keratin fibers; a.3) from 0.005% to 10% w / w of culture medium; where the pH of the medium is in the range from 7.5 to 8.5; b) keeping the mixture in a) at a temperature ranging from 20 ° C to 35 ° C and under stirring; c) separating the supernatant obtained in b); d) separating the hydrolyzed peptides from the supernatant from step c); Process according to Claim 1, characterized in that the bacterial concentration of item a.l) is 107 cells / ml. Process according to Claim 1, characterized in that the keratin fiber-containing material is selected from the group comprising hair, feathers, hooves, nails, horns and a mixture thereof. Process according to Claim 3, characterized in that the feathers are chosen from the group comprising feathers of chicken, chicken, ducks, geese and a mixture thereof. Process according to Claim 1, characterized in that the keratin fiber-containing material is at a concentration of 1% w / w. Process according to Claim 1, characterized in that the culture medium comprises yeast extract, peptone, KCl, sucrose or a mixture thereof. Process according to Claim 1, characterized in that the culture medium is at a concentration of 5%. Process according to Claim 1, characterized in that the pH is 8.0. Process according to Claim 1, characterized in that the temperature is 28 ° C. Process according to Claim 1, characterized in that the agitation is 300 rpm. Process according to Claim 1, characterized in that the separation mentioned in c) is carried out by centrifugation. Process according to Claim 1, characterized in that the separation mentioned in d) is carried out by means of ultrafiltration. Process according to Claim 1, characterized in that it comprises an additional step of preparing the keratin fiber-containing material before use in step a). Process according to Claim 13, characterized in that the additional step comprises one or more steps chosen from the group comprising: a) washing; b) drying; c) delipidation; and d) mixtures of a), b) and / or c). Process according to Claim 14, characterized in that the washing step is carried out with a suitable detergent. Process according to Claim 15, characterized in that the delipidation step comprises the use of a chloroform: methanol mixture in a ratio ranging from 0.5: 1 to 3: 1. 17 Keratin hydrolyzate, molecular weight 500Da to 100ODa, characterized in that it is obtained by the process described in claims 1 to 16.